Down-regulation of single-stranded DNA-binding protein 1 expression induced by HCMV infection promotes lipid accumulation in cells

The objective of this study was to observe the infection of human cytomegalovirus (HCMV) to human umbilical vein endothelial cells, and its effect on the expression of single-stranded DNA-binding protein (SSBP1) and on lipid metabolism in endothelial cells. We screened the differential expression of mRNAs after HCMV infection by suppression subtractive hybridization and the expression levels of SSBP1 mRNA and protein after HCMV infection by real-time PCR and western blot. After verification of successful infection by indirect immunofluorescent staining and RT-PCR, we found a differential expression of lipid metabolism-related genes including LDLR, SCARB, CETP, HMGCR, ApoB and LPL induced by HCMV infection. The expression levels of SSBP1 mRNA and protein after HCMV infection were significantly down-regulated. Furthermore, we found that upregulation of SSBP1 inhibited the expression of atherosclerosis-associated LDLR, SCARB, HMGCR, CETP as well as the accumulation of lipids in the cells. The results showed that the inhibition of SSBP1 by HCMV infection promotes lipid accumulation in the cells.

Cloning and characterization of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene from Paris fargesii Franch.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) plays an important role in catalyzing the first committed step of isoprenoids biosynthesis in mevalonic acid (MVA) pathway. Here, we cloned a full-length transcript of Paris fargesii Franch. The full-length cDNA of P. fargesii HMGR (Pf-HMGR, GenBank accession no. JX508638) was 1,973 bp and contained a 1,728 bp ORF encoding 576 amino acids. Sequence analysis revealed that the deduced Pf-HMGR had high similarity with HMGRs from other plants, including Ricinus communis (77%), Litchi chinensis (76%), Michelia chapensis (75%) and Panax quinquefolius (72%). It had a calculated molecular mass of about 62.13 kDa and an isoelectric point (pI) of 8.47. It contained two transmembrane domains, two putative HMGR binding sites and two NADP(H)-binding sites. The predicted 3-D structure revealed that Pf-HMGR had a similar spatial structure with other plant HMGRs. Three catalytic regions, including L-domain, N-domain and S-domain were detected by structural modeling of HMGR. Tissue expression analysis revealed that Pf-HMGR was strongly expressed in roots and stems than in leaves. Taken together, our data laid a foundation for further investigation of HMGR's functions and regulatory mechanisms in plants.

Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression

Background: The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome. Although the stimulation of diosgenin accumulation by ethylene has been reported in a few of plant species, its regulation is not yet characterized at the molecular level, the underlying molecular mechanism remains elusive. Results: In this study, the effects of ethylene on diosgenin biosynthesis in in vitro cultures of D. zingiberensis were described. The results showed that, in samples treated with ethylene at concentration E3 (10(4) dilution of 40% ethephon), the diosgenin biosynthesis was significantly promoted in comparison with the control samples. Treatment with high concentrations of ethylene had inhibitory effect, whereas with low concentration of the gas elicitor brought about no detectable deleterious effect on the growth rate and diosgenin content of the cultures. The considerable increase of diosgenin level in in vitro cultured Dioscorea zingiberensis by ethylene application is accompanied by the concomitant increase of soluble proteins and chlorophyll content. The gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase but not of squalene synthase or farnesyl pyrophosphate synthase were up-regulated by applied ethylene. Conclusions: Our results suggest that ethylene treatment enhanced diosgenin accumulation via up-regulation of the gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Human hydroxymethylglutaryl-coenzyme A reductase (HMGCR) and statin sensitivity.

While statins, hydroxymethylglutaryl-coenzyme A reductase (HMGCR) inhibitors, are clinically proven to reduce plasma cholesterol levels, a wide variation in inter-individual response to statin therapy has been observed. Pharmacogenetic studies have identified multiple loci that potentially contribute towards the statin response, including the HMGCR gene. To examine, if a statin-resistant, catalytically-active isoform of the human HMGCR could be generated, we have rationally altered the protein to include additional residues in the flap domain, which has a role in statin binding. Comparative enzyme assays with purified wild-type and mutant isoforms reveal the alteration imposes a slight (38%) decrease in the for the substrate, a near 2-fold increase in turnover number, and a 480% increase in the Ki for lovastatin. Thus, alterations in HMGCR could contribute towards the synergistic effects of multiple loci in the statin response.

Beneficial effects of a polar fraction of garlic (Allium sativum Linn) oil in rats fed with two different high fat diets.

Feeding a diet containing 20% of sesame oil (SO) or coconut oil (CNO) along with 2% cholesterol to rats for two months showed differences in their serum and tissue lipid profile and certain enzyme activities. Hyperlipidemia and related oxidative effects were more pronounced in coconut oil fed rats than those fed sesame oil. Feeding a combination of the oils (10% CNO +10% SO) lowered significantly the hyperlipidemia and certain other deleterious effects of CNO. Feeding a polar fraction of garlic oil (PFGO) prepared in the same way as for ajoene and administered at a dosage of 100 mg/kg along with each of the above oil containing diets counteracted significantly the hyperlipidemic, oxidant and also most of the other deleterious effects of the oils like raised lipid levels in serum and tissues, raised serum levels of AST and tissue levels of HMGCoA reductase and the lowered serum and tissue levels of glutathione reductase. The results support the claims that ajoene, the major polar compound of garlic oil, has very good biological action, which warrants further study.

Influence of coconut kernel protein on lipid metabolism in alcohol fed rats.

Male albino rats were given ethanol (3.76 g/kg body weight/day) to induce hyperlipidemia. The rats showed increased concentration of cholesterol and triglycerides in the serum and tissues. Inclusion of coconut protein and L-arginine into ethanol fed rats produced lower levels of total cholesterol, LDL+ VLDL cholesterol, triglycerides and atherogenic index in the serum. Concentration of tissue cholesterol and triglycerides was also lower in these groups. Administration of coconut protein and L-arginine in the ethanol fed rats caused decreased activity of HMG-CoA reductase in the liver and increased activity of lipoprotein lipase in the heart. The activities of malic enzyme and glucose-6-phosphate dehydrogenase were also lower in these groups. Feeding coconut protein and L-arginine in ethanol treated rats showed increased concentration of hepatic bile acids and fecal excretion of neutral sterols and bile acids. All these effects were comparable in rats fed coconut protein and those fed L-arginine. These observations indicate that the major factor responsible for the hypolipidemic effect of coconut protein is due to the high content of L-arginine.

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags.

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.

Impact of feeding ethanolic extracts of Achyranthes aspera Linn. on reproductive functions in male rats.

Feeding 50% ethanolic extract of A. aspera to male rats resulted in reduced sperm counts, weight of epididymis, serum level of testosterone and testicular activity of 3beta-hydroxysteroid dehydrogenase, while motility of the sperm and activity of the HMG CoA reductase were not affected. Cholesterol level in the testis, incorporation of labelled acetate into cholesterol, 17-ketosteroids in urine and hepatic and fecal bile acids were increased. The results suggest that ethanolic extract of A. aspera caused reproductive toxicity in male rats and the action may be by suppressing the synthesis of androgen.

Familial hypodigoxinemic membrane Na(+)-K(+) ATPase upregulatory syndrome - relation between digoxin status and cerebral dominance.

A family with coexistence of hypotension, recurrent respiratory infection, motor tics, obsessive compulsive disorder, major depressive disorder, early onset osteoporosis, low body mass index, bulimia nervosa and healthy aging with longevity is described. The family members had hyposexual behavior, less tendency for spirituality, had no insomnia but a tendency towards increased somnolence, no addictive behaviour, had more bonding and affectionate behavior and were less creative with an average IQ. There was no vascular thrombosis, systemic neoplasm and neuronal degeneration in the index family. All members of the family were left hemispheric dominant. The level of serum digoxin, HMG CoA reductase activity and dolichol was found to be decreased in all with a corresponding increase in RBC Na(+)-K(+) ATPase activity and serum ubiquinone magnesium level. There was increase in tyrosine catabolites and a reduction in tryptophan catabolites in serum. Total and individual glycosaminoglycan fractions, carbohydrate residues of glycoproteins, glycolipids, activity of GAG degrading enzymes and glycohydrolases were decreased in serum. The concentration of RBC membrane total GAG and carbohydrate residues of glycoproteins increased while cholesterol : phospholipid ratio of membrane decreased. The activity of free radical scavenging enzymes were increased while the concentration of free radicals decreased significantly. The same biochemical patterns were observed in left hemispheric dominance as opposed to right hemispheric dominance. The significance of these findings in the pathogenesis of these disorders is discussed.

Intestinal cholesterol synthesis and mobilisation to blood in sucrose-feeding and alcohol-intake.

Small intestines synthesize cholesterol to a greater extent than liver. Between starch-fed and sucrose-fed rats, using (14C) glucose, it was found that the synthesis of cholesterol by the jejunum of small intestines was greater in the sucrose-fed group than starch-fed group. By a novel experimental technique and using (14C) glucose, it was found that the contribution towards buffer representing lymph was greater in the sucrose-fed group (13.3%) than the controls (11%). Hypercholesterolemia on sucrose feeding may be at least partly due to contribution by the small intestines. Regarding alcohol, using (14C) glucose it was found that total synthesis of cholesterol by the small intestines was decreased in alcohol-fed rats. There was no difference in the cholesterol retained by the intestinal tissue between the controls and alcohol-fed animals while, the secretion towards buffer (lymph) was 9% as against 11. This indicates that there is contribution of cholesterol to blood from small intestines in alcohol-intake also but due to overall decrease in the intestinal synthesis of cholesterol, contribution of intestines to hypercholesterolemia may not be substantial as in the case of sucrose feeding. This is because in sucrose-feeding there is increased cholesterol synthesis.

Prenatal exposure of an alcoholic beverage (Arrack) on fetal lipid metabolism in rats.

The objective of this study was to determine the effects of a country liquor (Arrack) and the equivalent quantity of ethanol on liver function and lipid metabolism in utero. Female rats of average weight 125 g were exposed to Arrack (12 ml/kg body weight/day) and ethanol (3.2 ml/kg body weight/day) for 15 days before conception and throughout gestation. On 13th day and 19th day of gestation, altered liver function and hyperlipidemia was seen in the fetus of both the treated groups. Altered liver function was evidenced by the increased activity of alcohol dehydrogenase and glutamic pyruvic transaminase or alanine amino transferase (GPT). Hyperlipidemia was caused by increased biosynthesis since the incorporation of 14C acetate to lipids and activities of HMG CoA reductase and lipogenic enzymes were elevated. Arrack seemed to potentiate the toxicity induced by alcohol indicating the role of non ethanolic portion. Hepatic functions of the 13th day fetuses were effected to a lesser degree than the 19th day hepatic liver.

Effect of catechin on intestinal lipid metabolism.

On analysing the effect of catechin on intestinal lipid metabolism, an increase in the concentration of cholesterol in the duodenum and jejunum was observed along with an increase in the HMGCoA reductase activity. In the in vitro experiments also it was found that cholesterol and free fatty acid (FFA) levels were increased in these two regions. Binding of catechin with cholesterol in the lumen, reduces the availability of cholesterol for absorption which may in turn stimulate cholesterol biosynthesis and a rise in the HMGCoA reductase activity. These alterations produced by catechin may also be related to the degradation of cholesterol to bile acids, as endogenous cholesterol is the preferred substrate for bile acid synthesis.

Estatinas no tratamento da hipercolesterolemia / Statins in the hypercholesterolemia treatment

Estatinas säo drogas derivadas de microorganismos e que eficientemente interferem na síntese celular de colesterol por inibiçäo competitiva da enzima HMG-CoA-redutase. Näo obstante, as estatinas reduzem a colesterolemia por induzirem formaçäo de receptores que captam as LDL do plasma e por diminuirem a síntese de VLDL no fígado. Esta última explica o efeito parcial na queda da trigliceridemia. A eficiência das estatinas na diminuiçäo da colesterolemia é comparável à das resinas seqüestradoras de ácidos biliares, porém superios à dos fibrates e ácido nicotínico. Estatinas säo melhor toleradas do que estas últimas duas drogas, mas inferiores quanto à capacidade de diminuirem os triglicérides e de aumentarem o HDL-colesterol. Seletividade tissular varia entre as diversas estatinas, mas esta é uma questäo irrelevante tendo em vista a raridade dos efeitos colaterais. Conseqüentemente, estas drogas devem ser prescritas em razäo da potência farmacológica e do fator custo. Cinecoronarioangiografia seqüencial em pacientes com coronariopatia tratados com placebo em comparaçäo a estatinas isoladamente, indica que a doença arterial regride por métodos farmacológicos.

Impact of protein deficiency and exposure to hexachlorocyclohexane or malathion on lipid metabolism in pregnant rats.

Dietary intake of three oral doses of hexachlorocyclohexane (HCH) (60 mg/kg body wt) or malathion (500 mg/kg) by normal and protein-deficient diet fed pregnant rats on the 6th, 10th and 14th day of gestation resulted in the impairment of lipid metabolism, viz. hypercholesterolemia and hypertriglyceridemia. The cholesterol, triglyceride and phospholipid contents in serum, brain, liver, kidney and uterus were increased significantly by HCH and malathion exposure, irrespective of the protein content in the diet. The incorporation of [1,2-14C]acetate into the hepatic lipids was stimulated by both HCH and malathion, suggesting a higher rate of lipid synthesis in the liver of normal and protein-deficient diet fed dams. The low protein content in the diet intensified the pesticide-induced changes and more severe alterations were noticed in HCH exposed dams than in malathion exposed dams.

Avanços terapêuticos em cardiologia: parte 6; hipolipemiantes / Therapeutic development in cardiology

Os autores chamam a atençäo para a necessidade de as lipoproteínas LDL e VLDL serem reduzidas para níveis normais principalmente naqueles pacientes portadores de doença coronária ou que já submeteram-se à cirurgia de revascularizaçäo. Também enfatizam que ao lado da dieta, três säo as principais drogas usadas: gemfibrosil, para reduzir VLDL; inibidores da HMG GoA redutase para diminuir os níveis de LDL e o probucol como antioxidante

Dietary protein and cholesterol metabolism in small intestines.

Effect of quality and quantity of dietary protein on blood cholesterol and cholesterol metabolism in small intestines of rat was examined. Compared to casein, bengalgram in the diet decreased blood and intestinal cholesterol. It is suggested that this effect may be due to low levels of leucine in bengalgram protein and less release of insulin, an activator of HMG CoA Reductase. Low casein in the diet (12%) caused a decrease of blood and intestinal cholesterol while high casein in the diet (24%) caused a decrease of intestinal cholesterol only. It is suggested that both qualitywise and quantitywise, dietary protein influences body cholesterol. It is known that the nature and the quality of dietary fat and carbohydrates directly influence the metabolism of cholesterol. The dietary proteins might also have a significant role. Proteins are needed for the synthesis of enzymes required for the anabolism and catabolism of cholesterol and a few protein-aminoacids like leucine have a direct influence on cholesterol metabolism. Bengalgram was chosen for studying the effect of the quality of protein on cholesterol metabolism as there are reports in the literature that bengalgram feeding reduces blood cholesterol. Different amounts of casein-containing diet were used in a separate set of experiments to investigate the effects of low and high protein diets on cholesterol metabolism i.e. quantitative effects of dietary protein. For many years, liver was enjoying the privilege of maintaining the homeostasis of blood cholesterol. Recently it has been shown that small intestines also synthesises considerable amounts of cholesterol. Hence, cholesterol metabolism was investigated separately in duodenum, jejunum and ileum.

Effect of antimicrobials on cholesterol synthesis and content in liver and small intestines.

The antimicrobials tetracycline, ampicillin and bactrim (cotrimoxazole) decreased HMG CoA reductase activity in liver and small intestines of albino rats. Diminished incorporation of 1, 2, 14C acetate into cholesterol of small intestines in bactrim group was noted. There was a significant fall in cholesterol content of liver, duodenum, jejunum and ileum of the bactrim group and jejunum only in tetracycline group.